A Study On Intratumoral Heterogeneity And Immune Microenvironment In Liver Cancer Based On Multi-omics Data

The First PartIntegrated multi-omic analysis reveals comprehensive tumor heterogeneity and novel immunophenotypic classification in hepatocellular carcinomas BackgroundHepatocellular carcinoma(HCC)is heterogeneous,especially in multifocal tumors,which decreases the efficacy of clinical treatments.Understanding intratumoral and intertumoral heterogeneity in different dimensions from the genome to the phenome is critical when developing novel treatment strategies.However,a comprehensive investigation of tumor heterogeneity in HCC is lacking,and the available evidence regarding tumor heterogeneity has not led to improvements in clinical practice.AimThis study aims to integrate the information of tumor genome,expression profile and phenotypes,and to provide a landscape of intratumoral heterogeneity in HCC based on multi-omic data.The rule of intratumoral heterogeneity from genome to phenotype would be described to accelerate the discovery of the best intervention dimension and strategy which can effectively minimize the impact of intratumoral heterogeneity.Methods and MaterialsWe harvested 42 samples from eight HCC patients and evaluated tumor heterogeneity using whole-exome sequencing(WES),bulk RNA sequencing,mass spectrometry-based proteomics and metabolomics,as well as cytometry by time-of-flight(Cy TOF).Immunohistochemistry and quantitative polymerase chain reactions(q PCR)were performed to confirm the expression levels of genes.ResultsThis multi-omics study showed that tumor heterogeneity is considerable with regard to the genomes,transcriptomes,proteomes,and metabolomes of lesions and tumors.The immune status of the HCC microenvironment had a relatively low level of heterogeneity.We found that targeting local immunity could be a suitable intervention with balanced precision and practicability.By clustering immune cells in the HCC microenvironment,we identified three distinctive HCC subtypes with immunocompetent,immunodeficient,and immunosuppressive features.We further revealed the specific metabolic features and cytokine/chemokine expression levels of the different subtypes.In addition,we confirmed that determining the expression levels of PTPRC and FOXP3 using quantitative PCR and immunohistochemistry in two independent HCC cohorts facilitated the correct classification of HCC patients and the prediction of their prognosis.ConclusionWe have revealed that there is comprehensive intratumoral and intertumoral heterogeneity in all dimensions of HCC.Based on the results,we propose a novel immunophenotypic classification of HCCs that facilitates prognostic prediction and may support decision making with regard to the choice of therapy.The Second PartHeterogeneous immune cell infiltration reveals tumor-immune regulatory mechanisms in hepatocellular carcinomasBackgroundImmunotherapy is a promising strategy for treating hepatocellular carcinoma(HCC),but the current response rate of such strategy is low.Tumor-immune ecosystem is pivotal for effective immunotherapy and it varies among patients with HCC,yet its underlying regulatory mechanisms are poorly described.AimThis study aims to offer the landscape of tumor infiltrating immune cells systematically and unearth the interaction between immune cell subtypes.The key regulatory mechanism of tumor-immune ecosystem heterogeneity in HCC will be explored using tumor expression profile,so as to provide a novel therapeutic target for enhanced immunotherapy.Methods and MaterialsHCC samples were analyzed using mass cytometry,RNA sequencing,and immunohistochemistry to establish the mechanisms of tumor-immune interactions.An integrated analysis of immune cell subtypes,tumor transcriptome,and clinical parameters was performed.In vitro,ex vivo and in vivo mouse model experiments were conducted to confirm the findings.ResultsAn in-depth analysis of the infiltrating immune cells in HCC facilitated the identification of 48 immune cell subsets based on their phenotypic properties.A phenotype-based trajectory inference indicated distinct states of immune cell exhaustion.Function-associated communities were constructed based on the correlations between immune clusters.PD-L1+ plasma cells of the exhausted community demonstrated immunosuppressive ability by releasing secretory PD-L1,which predicted prognosis of patients with HCC.This study revealed a comprehensive delineation of the immunoregulation of tumor-related pathways such as the Wnt/β-catenin signaling pathway.Furthermore,fatty acids metabolism was identified as a key contributor of T cell dysfunction.Overexpression of GGT5 and injection of leukotriene D4 promoted recovery of T cells from exhaustion.ConclusionThis study presented a heterogeneous landscape of tumor-immune ecosystem in HCC.PD-L1+ plasma cells can be potential biomarkers for patient survival,and arachidonic acid metabolism can be a potential target for enhanced immunotherapy in HCC.